RESUMO
BACKGROUND: Macrophages release not only cytokines but also extracellular vesicles (EVs). which are small membrane-derived nanovesicles with virus-like properties transferring cellular material between cells. Until now, the consequences of macrophage plasticity on the release and the composition of EVs have been poorly explored. In this study, we determined the impact of high-glucose (HG) concentrations on macrophage metabolism, and characterized their derived-EV subpopulations. Finally, we determined whether HG-treated macrophage-derived EVs participate in immune responses and in metabolic alterations of skeletal muscle cells. METHODS: THP1-macrophages were treated with 15mM (MG15) or 30mM (MG30) glucose. Then, M1/M2 canonical markers, pro- and anti-inflammatory cytokines, activities of proteins involved in glycolysis or oxidative phosphorylation were evaluated. Macrophage-derived EVs were characterized by TEM, NTA, MRSP, and 1H-Nuclear magnetic resonance spectroscopy for lipid composition. Macrophages or C2C12 muscle cells were used as recipients of MG15 and MG30-derived EVs. The lipid profiles of recipient cells were determined, as well as proteins and mRNA levels of relevant genes for macrophage polarization or muscle metabolism. RESULTS: Untreated macrophages released small and large EVs (sEVs, lEVs) with different lipid distributions. Proportionally to the glucose concentration, glycolysis was induced in macrophages, associated to mitochondrial dysfunction, triacylglycerol and cholesterol accumulation. In addition, MG15 and MG30 macrophages had increased level of CD86 and increase release of pro-inflammatory cytokines. HG also affected macrophage sphingolipid and phospholipid compositions. The differences in the lipid profiles between sEVs and lEVs were abolished and reflected the lipid alterations in MG15 and MG30 macrophages. Interestingly, MG15 and MG30 macrophages EVs induced the expression of CD163, Il-10 and increased the contents of triacylglycerol and cholesterol in recipient macrophages. MG15 lEVs and sEVs induced insulin-induced AKT hyper-phosphorylation and accumulation of triacylglycerol in myotubes, a state observed in pre-diabetes. Conversely, MG30 lEVs and sEVs induced insulin-resistance in myotubes. CONCLUSIONS: As inflammation involves first M1 macrophages, then the activation of M2 macrophages to resolve inflammation, this study demonstrates that the dialog between macrophages through the EV route is an intrinsic part of the inflammatory response. In a hyperglycemic context, EV macrophages could participate in the development of muscle insulin-resistance and chronic inflammation.
Assuntos
Vesículas Extracelulares , Insulinas , Humanos , Macrófagos/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Vesículas Extracelulares/metabolismo , Lipídeos , Homeostase , Triglicerídeos/metabolismo , Colesterol/metabolismo , Insulinas/metabolismoRESUMO
BACKGROUND: Adipose tissue is infiltrated with various immune cells, including Th17 lymphocytes and monocytes/macrophages, in obese individuals. We have previously demonstrated the role of obese adipose-derived stem cells (ob-ASC) and adipocytes (AD) in the mediation of inflammation through promotion of Th17 cells and activation of monocytes. Such an inflammation resulted in impaired ob-ASC adipogenesis and AD insulin response. In the present study, we investigated the role of IL-17A in the impairment of these functions. METHODS: With this aim, we used Secukinumab, a potent human anti-IL17A monoclonal antibody which has been approved for the treatment of some IL-17A related inflammatory diseases, notably Psoriasis. This antibody was added or not to phytohemagglutinin A-activated co-cultures of ob-ASC and mononuclear cells. The conditioning media of those co-cultures were harvested and added to AD ongoing differentiation from ob-ASC. Adipogenesis, insulin sensitivity and secretion of inflammatory cytokines were then measured using qRT-PCR, Western blots and ELISAs, respectively. RESULTS: Surprisingly, we did not observe any direct effect of IL-17A on ob-ASC adipogenesis, despite sensitivity of ob-ASC to IL-17A. Moreover, IL-17A blockade, with the help of Secukinumab, did not lead to the recovery of adipogenesis and insulin response, when these functions were impaired by the presence of an inflammatory conditioning medium. However, the up-regulation of IL6 and IL1B mRNA expression by AD submitted to inflammatory conditioning medium was inhibited in the presence of Secukinumab, which indicates that IL-17A may play a role in the propagation of inflammation towards AD. IN CONCLUSION: we show herein that IL-17A does not play a major role in the impairment of adipogenesis and/or insulin resistance mediated by an inflammatory environment, but contributes to the propagation of inflammation in human obese adipose tissues. This suggests a beneficial effect of anti-IL17A mAb in inflammatory pathologies, where obesity contributes to poorer response to biologic treatments.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Anticorpos Monoclonais Humanizados/farmacologia , Interleucina-17/antagonistas & inibidores , Células-Tronco Mesenquimais/efeitos dos fármacos , Obesidade/metabolismo , Adipócitos/metabolismo , Adipogenia/genética , Tecido Adiposo , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Citocinas/genética , Citocinas/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Resistência à Insulina/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Células-Tronco Mesenquimais/metabolismo , Monócitos/efeitos dos fármacos , Obesidade/genética , Fito-Hemaglutininas/farmacologiaRESUMO
Minor histocompatibility (H) Ags are classically described as self-peptides derived from intracellular proteins that are expressed at the cell surface by MHC class I and class II molecules and that induce T cell alloresponses. We have isolated three different T cell populations from a skin biopsy of a patient suffering from acute graft-versus-host disease following sex-mismatched HLA-identical bone marrow transplantation. The first population was: 1) CD4(+)/CD8(+) double-positive; 2) specific for an HLA class I-restricted autosomal Ag; 3) expressed a Tr1 profile with high levels of IL-10, but low IL-2 and IFN-γ; and 4) exerted regulatory function in the presence of recipient APCs. The second was CD8 positive, specific for an HLA class I-restricted autosomally encoded minor H Ag, but was only weakly cytotoxic. The third was CD4 single positive, specific for an HLA-DR7-restricted HY epitope and exerted both proliferative and cytotoxic functions. Identification of the peptide recognized by these latter cells revealed a new human HY epitope, TGKIINFIKFDTGNL, encoded by RPS4Y and restricted by HLA-DR7. In this paper, we show human CD4/CD8 double-positive, acute graft-versus-host disease-protective, minor H Ag-specific regulatory T cells and identify a novel HLA-DR7/ HY T cell epitope, encoded by RPS4Y, a potential new therapeutic target.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Antígeno H-Y/imunologia , Antígeno HLA-DR7/genética , Antígenos de Histocompatibilidade Menor/imunologia , Linfócitos T Reguladores/imunologia , Apresentação de Antígeno/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Transformada , Separação Celular/métodos , Células Clonais , Feminino , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Cadeias HLA-DRB1/genética , Humanos , Masculino , Antígenos de Histocompatibilidade Menor/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismoRESUMO
Obesity is often associated with adipose tissue (AT) inflammation and immune cell infiltration. Writing recently in Cell Reports, Liao et al. investigated the mechanisms of T cell infiltration of AT using single cell (sc)RNA-sequencing (RNA-seq), transplantation studies, in vitro co-cultures, and knock-out mice. They highlighted the crucial role of C-C motif chemokine ligand 5 (CCL5)-secreting adipose stem cells (ASCs), offering insights for potential therapies.
Assuntos
Medula Óssea/patologia , Quimerismo , Doença Enxerto-Hospedeiro/etiologia , Transplante de Órgãos/efeitos adversos , Vísceras/transplante , Evolução Fatal , Feminino , Histocompatibilidade/imunologia , Humanos , Pessoa de Meia-Idade , Transplante de Órgãos/métodos , Rituximab/uso terapêutico , Linfócitos T/imunologia , Doadores de TecidosRESUMO
OBJECTIVE: To investigate the production of type I interferon (IFN) by myoblasts and to identify its cell source and the link to Toll-like receptor (TLR) and C-type lectin receptor (CLR) expression and function in myositis biopsy sections. METHODS: Production of IFNß was assessed in cultured myoblasts after stimulation with the TLR-3 agonist poly(I-C) or with cytokines involved in Th1 and Th17 differentiation. Expression of HLA class I molecules by myoblasts was analyzed by fluorescence-activated cell sorting after activation of TLR-3 and IFNß neutralization. In muscle biopsy samples from patients with polymyositis or dermatomyositis, expression of IFNß, CD56 (a marker of immature muscle precursors), and HLA class I was analyzed using immunohistochemistry. Inflammatory infiltrates were characterized for the expression of myeloid dendritic cells (DCs), their associated CLRs, and the products of activated DCs, interleukin-12 (IL-12), and IL-23. RESULTS: In cultured myoblasts, stimulation of TLR-3 induced the production of IFNß when combined with IFNγ and up-regulated the expression of HLA class I molecules, which was decreased after IFNß blockade. In myositis biopsy tissues, immature muscle precursors overexpressing HLA class I were identified as a source of IFNß. CLRs associated with myeloid DCs were broadly expressed in inflammatory infiltrates, in association with IL-12 and IL-23, and with immature muscle precursors. CONCLUSION: Immature muscle precursors may represent a local source of IFNß and the target of an immune response involving activated DCs associated with the expression of CLRs and of IL-12 and IL-23, which are implicated in T cell polarization. In turn, such local production of IFNß after TLR-3 activation in the presence of the Th1 cytokine IFNγ may explain HLA class I overexpression in myositis.
Assuntos
Dermatomiosite/metabolismo , Genes MHC Classe I/fisiologia , Interferon beta/metabolismo , Mioblastos/metabolismo , Polimiosite/metabolismo , Receptor 3 Toll-Like/metabolismo , Adulto , Idoso , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Regulação para CimaRESUMO
OBJECTIVE: Th17 cells have been implicated in rheumatoid arthritis (RA). We hypothesized that the interaction of T cells with bone marrow-derived mesenchymal stem cells (BM-MSCs) or with fibroblast- like synoviocytes (FLS) might, with the help of T cell-secreted inflammatory cytokines (i.e., interleukin-17A [IL-17A], tumor necrosis factor α [TNFα], and/or interferon-γ [IFNγ]), promote Th17 cell expansion and activation. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy blood donors were cocultured with BM-MSCs or FLS from RA patients or osteoarthritis (OA) patients. Cocultures were exposed to phytohemagglutinin with or without IL-17A, TNFα, or IFNγ. Quantitative reverse transcription-polymerase chain reaction analysis, enzyme-linked immunosorbent assay, and cytofluorometry were used to measure IL-17A production. RESULTS: Interaction of PBMCs with BM-MSCs inhibited Th1 and Th2 responses, but promoted Th17 cell expansion, as early as 24 hours, as demonstrated by increases in retinoic acid receptor-related orphan nuclear receptor γ or IL-17A messenger RNA (mRNA) levels, IL-17A secretion levels, and IL-17A-secreting cell frequency, as well as by T cell switching to the Th17 pathway after 2 rounds of stimulation with MSCs. IL-17A production was also increased in PBMCs stimulated with anti-CD3 plus anti-CD28 or in isolated CD3+ or CD45RO+ T cells, thus demonstrating the role of T cell activation. Levels of mRNA for IL-6, IL-8, and IL-1ß were further amplified when T cell-secreted inflammatory cytokines were added. Interestingly, OA FLS or RA FLS also enhanced IL-17A and IL-6 production, but only RA FLS enhanced IFNγ and IL-1ß production. We further demonstrated that MSC-mediated Th17 promotion requires caspase 1 activation by using an inhibitory peptide and measuring its activity. CONCLUSION: We found that the interaction of MSCs or FLS with T cells promotes the activation and expansion of Th17 cells through caspase 1 activation. Since proinflammatory and T cell-secreted inflammatory cytokines are also amplified, this mechanism may participate in the chronicity of RA.
Assuntos
Artrite Reumatoide/metabolismo , Células da Medula Óssea/metabolismo , Caspase 1/metabolismo , Células-Tronco Mesenquimais/metabolismo , Membrana Sinovial/metabolismo , Células Th17/metabolismo , Artrite Reumatoide/patologia , Células da Medula Óssea/patologia , Células Cultivadas , Humanos , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Células-Tronco Mesenquimais/patologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Membrana Sinovial/patologia , Regulação para CimaRESUMO
Adipose tissue-derived mesenchymal stem cells (ASCs) are adult stem cells, endowed with self-renewal, multipotent capacities, and immunomodulatory properties, as mesenchymal stem cells (MSCs) from other origins. However, in a pathological context, ASCs like MSCs can exhibit pro-inflammatory properties and attract inflammatory immune cells at their neighborhood. Subsequently, this creates an inflammatory microenvironment leading to ASCs' or MSCs' dysfunctions. One such example is given by obesity where adipogenesis is impaired and insulin resistance is initiated. These opposite properties have led to the classification of MSCs into two categories defined as pro-inflammatory ASC1 or anti-inflammatory ASC2, in which plasticity depends on the micro-environmental stimuli. The aim of this review is to (i) highlight the pathogenic role of ASCs during obesity and obesity-related inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, and cancer; and (ii) describe some of the mechanisms leading to ASCs dysfunctions. Thus, the role of soluble factors, adhesion molecules; TLRs, Th17, and Th22 cells; γδ T cells; and immune checkpoint overexpression will be addressed.
Assuntos
Artrite Reumatoide , Células-Tronco Mesenquimais , Humanos , Tecido Adiposo , Obesidade/patologia , ImunomodulaçãoRESUMO
BACKGROUND: Obesity is a well-known risk factor for cancer. We have previously reported the role of adipose-tissue-derived mesenchymal stem cells from obese individuals (ob-ASC) in the promotion of pathogenic Th17 cells and immune check point (ICP) upregulation. Thus, we postulated herein that this mechanism could contribute to breast cancer (BC) aggressiveness. METHODS: Conditioning medium (CM) from mitogen-activated ob-ASC and immune cell co-cultures were added to two human breast cancer cell line (BCCL) cultures. Expressions of pro-inflammatory cytokines, angiogenesis markers, metalloproteinases, and PD-L1 (a major ICP) were measured at the mRNA and/or protein levels. BCCL migration was explored in wound healing assays. Anti-cytokine neutralizing antibodies (Ab) were added to co-cultures. RESULTS: CM from ob-ASC/MNC co-cultures increased IL-1ß, IL-8, IL-6, VEGF-A, MMP-9, and PD-L1 expressions in both BCCLs and accelerated their migration. The use of Abs demonstrated differential effects for IL-17A and IFNγ on BCCL pro-inflammatory cytokine over-expression or PD-L1 upregulation, respectively, but potentiating effects on BCCL migration. Finally, co-cultures with ob-ASC, but not lean ASC, enhanced PD-L1 expression. CONCLUSIONS: Our results demonstrate increased inflammation and ICP markers and accelerated BCCL migration following the activation of pathogenic Th17 cells by ob-ASC, which could represent a new mechanism linking obesity with BC progression.
RESUMO
The discovery of leptin in the 1990s led to a reconsideration of adipose tissue (AT) as not only a fatty acid storage organ, but also a proper endocrine tissue. AT is indeed capable of secreting bioactive molecules called adipokines for white AT or batokines for brown/beige AT, which allow communication with numerous organs, especially brain, heart, liver, pancreas, and/or the vascular system. Adipokines exert pro or anti-inflammatory activities. An equilibrated balance between these two sets ensures homeostasis of numerous tissues and organs. During the development of obesity, AT remodelling leads to an alteration of its endocrine activity, with increased secretion of pro-inflammatory adipokines relative to the anti-inflammatory ones, as shown in the graphical abstract. Pro-inflammatory adipokines take part in the initiation of local and systemic inflammation during obesity and contribute to comorbidities associated to obesity, as detailed in the present review.
Assuntos
Adipocinas , Obesidade , Humanos , Tecido Adiposo , Tecido Adiposo Marrom , Tecido Adiposo Branco , FígadoRESUMO
Arterial allograft represents a material of choice for primary arterial revascularization in liver transplantation (LT) when interposition of a vascular conduit is required. In case of non-availability of such graft, the use of cryopreserved vessels should be an interesting option. Three patients were grafted using a cryopreserved iliac artery allograft (CIAA) previously harvested and stored at -140°C in a tissue bank. An auxiliary partial LT was performed in one patient for acute liver failure. During follow-up, an efficient regeneration of the native hemi-liver was observed while atrophy of the auxiliary graft occurred, leading to functional portal vein and hepatic artery thrombosis at six and nine months, respectively. Two other patients presented with celiac trunk compression because of arcuate ligament without available arterial allograft in the donor. Late arterial thrombosis occurred at six months in one patient without impairment of graft function. The last patient was alive and symptom free 29 months after LT with a patent cryopreserved arterial conduit. Our preliminary results suggest that CIAA might represent an efficient solution as vessel interposition for primary arterial hepatic revascularization in LT setting when no other suitable graft is available. However, long-term patency of CIAA remains questionable.
Assuntos
Criopreservação , Rejeição de Enxerto/etiologia , Artéria Hepática/fisiopatologia , Artéria Ilíaca/transplante , Transplante de Fígado , Fígado/irrigação sanguínea , Transplante Homólogo , Adulto , Anastomose Cirúrgica , Feminino , Humanos , Artéria Ilíaca/cirurgia , Fígado/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Procedimentos Cirúrgicos VascularesRESUMO
IFN-gamma has been shown to inhibit monocyte (Mo) differentiation into mature dendritic cells (DC). Because IFN-gamma also plays a role in tolerance induction, we asked whether this could be related to generation of tolerogenic DC (Tol-DC). Toward this aim, we cultured Mo with GM-CSF plus IL-4 in the presence or absence of IFN-gamma for 6 days and induced their maturation with TNF-alpha for 2 additional days. We showed that IFN-gamma deviated Mo differentiation from mature DC toward Tol-DC. Indeed, IFN-gamma-generated DC 1) expressed moderate levels of costimulatory molecules, but high levels of Langerin and CD123 molecules, 2) were maturation resistant, and 3) were unable to efficiently present alloantigen to T cells. More interestingly, naive CD4(+) T cells primed with IFN-gamma-generated DC expressed FoxP3 mRNA at high levels and exerted regulatory functions upon secondary stimulation with alloantigen. To address whether endogenously secreted IFN-gamma mediates a similar effect, we used the alloreaction as a model. We showed that cell-free supernatant harvested from an HLA-mismatched, but not HLA-identical, alloresponse induced differentiation of Mo into Tol-DC able to promote regulatory T cell generation. Moreover, when supplemented with GM-CSF plus IL-4, HLA-mismatched cell-free supernatant inhibited differentiation of Mo into mature DC. Finally, by adding Abs directed against inflammatory cytokines, we demonstrated that IFN-gamma plays a preponderant role in this inhibition. In conclusion, our results clearly demonstrate that exogenous or endogenous IFN-gamma, as well, induces differentiation of Mo toward Tol-DC, which results in FoxP3(+) regulatory T cell promotion.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/biossíntese , Tolerância Imunológica , Interferon gama/metabolismo , Monócitos/imunologia , Linfócitos T Reguladores/imunologia , Sistema Livre de Células/imunologia , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Humanos , Imunofenotipagem , Interferon gama/fisiologia , Interleucina-4/fisiologia , Teste de Cultura Mista de Linfócitos , Monócitos/citologia , Linfócitos T Reguladores/citologiaRESUMO
The PD-L1/PD-1 immune checkpoint axis is the strongest T cell exhaustion inducer. As immune dysfunction occurs during obesity, we analyzed the impact of obesity on PD-L1/PD-1 expression in white adipose tissue (WAT) in mice and in human white adipocytes. We found that PD-L1 was overexpressed in WAT of diet-induced obese mice and was associated with increased expression of PD-1 in visceral but not subcutaneous WAT. Human in vitro cocultures with adipose-tissue-derived mesenchymal stem cells (ASC) and mononuclear cells demonstrated that the presence of ASC harvested from obese WAT (i) enhanced PD-L1 expression as compared with ASC from lean WAT, (ii) decreased Th1 cell cytokine secretion, and (iii) resulted in decreased cytolytic activity towards adipocytes. Moreover, (iv) the implication of PD-L1 in obese ASC-mediated T cell dysfunction was demonstrated through PD-L1 blockade. Finally, (v) conditioned media gathered from these cocultures enhanced PD-L1 expression in freshly differentiated adipocytes, depending on IFNγ. Altogether, our results suggest that PD-L1 is overexpressed in the WAT of obese individuals during IFNγ secretion, leading to T cell dysfunction and notably reduced cytolytic activity. Such a mechanism could shed light on why adipose-tissue-infiltrating viruses, such as SARS-CoV-2, can worsen disease in obese individuals.
Assuntos
Tecido Adiposo Branco/metabolismo , Antígeno B7-H1/biossíntese , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Obesidade/metabolismo , Linfócitos T/imunologia , Animais , COVID-19/imunologia , Diferenciação Celular , Técnicas de Cocultura , Humanos , Imuno-Histoquímica , Inflamação , Interferon gama/metabolismo , Leucócitos Mononucleares/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/imunologia , SARS-CoV-2 , Linfócitos T/citologiaRESUMO
Taking advantage of the wide tropism of baculoviruses (BVs), we constructed a recombinant BV (BV(CAR)) pseudotyped with human coxsackie B-adenovirus receptor (CAR), the high-affinity attachment receptor for adenovirus type 5 (Ad5), and used the strategy of piggybacking Ad5-green fluorescent protein (Ad5GFP) vector on BV(CAR) to transduce various cells refractory to Ad5 infection. We found that transduction of all cells tested, including human primary cells and cancer cell lines, was significantly improved using the BV(CAR)-Ad5GFP biviral complex compared to that obtained with Ad5GFP or BV(CAR)GFP alone. We determined the optimal conditions for the formation of the complex and found that a high level of BV(CAR)-Ad5GFP-mediated transduction occurred at relatively low adenovirus vector doses, compared with transduction by Ad5GFP alone. The increase in transduction was dependent on the direct coupling of BV(CAR) to Ad5GFP via CAR-fiber knob interaction, and the cell attachment of the BV(CAR)-Ad5GFP complex was mediated by the baculoviral envelope glycoprotein gp64. Analysis of the virus-cell binding reaction indicated that the presence of BV(CAR) in the complex provided kinetic benefits to Ad5GFP compared to the effects with Ad5GFP alone. The endocytic pathway of BV(CAR)-Ad5GFP did not require Ad5 penton base RGD-integrin interaction. Biodistribution of BV(CAR)-Ad5Luc complex in vivo was studied by intravenous administration to nude BALB/c mice and compared to Ad5Luc injected alone. No significant difference in viscerotropism was found between the two inocula, and the liver remained the preferred localization. In vitro, coagulation factor X drastically increased the Ad5GFP-mediated transduction of CAR-negative cells but had no effect on the efficiency of transduction by the BV(CAR)-Ad5GFP complex. Various situations in vitro or ex vivo in which our BV(CAR)-Ad5 duo could be advantageously used as gene transfer biviral vector are discussed.
Assuntos
Adenovírus Humanos/genética , Baculoviridae/genética , Vetores Genéticos , Receptores Virais/genética , Transdução Genética , Adenovírus Humanos/ultraestrutura , Animais , Baculoviridae/ultraestrutura , Linhagem Celular , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Feminino , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Recombinantes de Fusão/genéticaRESUMO
BACKGROUND: Advanced glycation end products (AGE) are a marker of various diseases including diabetes, in which they participate to vascular damages such as retinopathy, nephropathy and coronaropathy. Besides those vascular complications, AGE are involved in altered metabolism in many tissues, including adipose tissue (AT) where they contribute to reduced glucose uptake and attenuation of insulin sensitivity. AGE are known to contribute to type 1 diabetes (T1D) through promotion of interleukin (IL)-17 secreting T helper (Th17) cells. AIM: To investigate whether lean adipose-derived stem cells (ASC) could be able to induce IL-17A secretion, with the help of AGE. METHODS: As we have recently demonstrated that ASC are involved in Th17 cell promotion when they are harvested from obese AT, we used the same co-culture model to measure the impact of glycated human serum albumin (G-HSA) on human lean ASC interacting with blood mononuclear cells. IL-17A and pro-inflammatory cytokine secretion were measured by ELISA. Receptor of AGE (RAGE) together with intercellular adhesion molecule 1 (ICAM-1), human leukocyte Antigen (HLA)-DR, cluster of differentiation (CD) 41, and CD62P surface expressions were measured by cytofluorometry. Anti-RAGE specific monoclonal antibody was added to co-cultures in order to evaluate the role of RAGE in IL-17A production. RESULTS: Results showed that whereas 1% G-HSA only weakly potentiated the production of IL-17A by T cells interacting with ASC harvested from obese subjects, it markedly increased IL-17A, but also interferon gamma and tumor necrosis factor alpha production in the presence of ASC harvested from lean individuals. This was associated with increased expression of RAGE and HLA-DR molecule by co-cultured cells. Moreover, RAGE blockade experiments demonstrated RAGE specific involvement in lean ASC-mediated Th-17 cell activation. Finally, platelet aggregation and ICAM-1, which are known to be induced by AGE, were not involved in these processes. CONCLUSION: Thus, our results demonstrated that G-HSA potentiated lean ASC-mediated IL-17A production in AT, suggesting a new mechanism by which AGE could contribute to T1D pathophysiology.
RESUMO
SCOPE: Obese adipose tissue (AT) is infiltrated by inflammatory immune cells including IL-17A-producing-T (Th17) cells. It has been previously demonstrated that adipose-derived stem cells from obese (ob-ASCs), but not lean AT promote Th17 cells. Because n-3 PUFAs are known to inhibit obese AT inflammation, it is tested here whether they could inhibit ob-ASC-mediated IL-17A secretion. METHODS AND RESULTS: The n-3 PUFA precursor, alpha-linolenic acid (ALA), or its derivatives, eicosapentaenoic, or docosahexaenoic acid, is added to co-cultures of human ob-ASCs and mononuclear cells (MNCs). All three inhibited IL-17A, but not IL-1ß, IL-6, nor TNFα secretion. As a control, palmitic acid (PA), a saturated fatty acid, did not inhibit IL-17A secretion. ALA also inhibited IL-17A secretion mediated by adipocytes differentiated from ob-ASCs. Toll-like-receptor 4 is shown to be involved in ob-ASC-mediated-IL-17A secretion, and to be inhibited by ALA, together with Cyclo-Oxygenase-2 and Signal-Transducer-and-Activator-of-transcription-3. In addition, ALA down-regulated Intercellular-Adhesion-Molecule-1 (ICAM-1) expression in both monocytes and ASCs, which resulted in decreased interactions between ob-ASCs and MNCs, and inhibition of IL-17A secretion. CONCLUSION: It is demonstrated herein that ALA inhibits Th17 cell promotion, through decreased ICAM-1expression in both ob-ASCs and monocytes. This novel mechanism may contribute to explain the beneficial effects of n-3 PUFA in IL-17A-related inflammatory pathologies.
Assuntos
Tecido Adiposo/citologia , Ácidos Graxos Ômega-3/farmacologia , Molécula 1 de Adesão Intercelular/genética , Interleucina-17/antagonistas & inibidores , Obesidade/metabolismo , Células-Tronco/fisiologia , Células Th17/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Técnicas de Cocultura , Humanos , Interleucina-17/biossíntese , Fator de Transcrição STAT3/antagonistas & inibidores , Células-Tronco/efeitos dos fármacos , Células-Tronco/imunologia , Células Th17/imunologia , Receptor 4 Toll-Like/antagonistas & inibidores , Ácido alfa-Linolênico/farmacologiaRESUMO
As a result of their potent antigen-presentation function, dendritic cells (DC) are important tools for cell therapy programs. In vitro-generated DC from enriched CD34+ hematopoietic stem cells (HSC; enriched CD34 DC) have already proven their efficiency in Phase I/II clinical trials. Here, we investigated whether enrichment of CD34+ HSC before the onset of culture was absolutely required for their differentiation into DC. With this aim, we developed a new two-step culture method. PBMC harvested from G-CSF-mobilized, healthy patients were expanded for 7 days during the first step, with early acting cytokines, such as stem cell factor, fetal liver tyrosine kinase 3 ligand (Flt-3L), and thrombopoietin. During the second step, expanded cells were then induced to differentiate into mature DC in the presence of GM-CSF, Flt-3L, and TNF-alpha for 8 days, followed by LPS exposure for 2 additional days. Our results showed that the rate of CD34+/CD38+/lineageneg cells increased 19.5+/-10-fold (mean+/-sd) during the first step, and the expression of CD14, CD1a, CD86, CD80, and CD83 molecules was up-regulated markedly following the second step. When compared with DC generated from enriched CD34+ cells, which were expanded for 7 days before differentiation, DC derived from nonenriched peripheral blood stem cells showed a similar phenotye but higher yields of production. Accordingly, the allogeneic stimulatory capacity of the two-step-cultured DC was as at least as efficient as that of enriched CD34 DC. In conclusion, we report herein a new two-step culture method that leads to high yields of mature DC without any need of CD34+ HSC enrichment.
Assuntos
Antígenos CD34/metabolismo , Células Dendríticas/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Células-Tronco Hematopoéticas/fisiologia , Técnicas de Cultura de Tecidos/métodos , Antígenos de Diferenciação/análise , Diferenciação Celular , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/fisiologia , Proteínas RecombinantesRESUMO
OBJECTIVE: Bone marrow mesenchymal stem cells (MSC) participate in the bone marrow microenvironment by providing growth factors and matrix proteins, which play a role in the regulation of hematopoiesis, through cell-to-cell interactions. Recently, MSC have been demonstrated to improve expansion of cord blood heamtopoietic stem cells (HSC). METHODS: In this report, we evaluated the impact of MSC on ex vivo expansion of adult mobilized peripheral blood stem cells (PBSC). Moreover, the effect of MSC on the expanded PBSC allostimulatory capacity was also investigated, due to the well-known immunomodulatory properties of MSC. In addition, the requirement for cell-cell contact in this MSC coculture system was investigated using a transwell system. RESULTS: Our results show that MSC greatly improved the expansion rate of adult PBSC cells relative to the absolute number of 1) clonogenic cells, 2) long-term cultured cells, or 3) CD34(+) cells. Whereas high levels of IL-6 on its own was sufficient to significantly improve PBSC expansion, direct contact between MSC and PBSC was required to achieve maximal expansion. Finally, MSC decreased the allostimulatory capacity of expanded PBSC. CONCLUSION: Our data show that MSC efficiently improve expansion of adult PBSC, together with decreasing their allostimulatory capacity. Therefore, this study should provide a clinically relevant method for optimizing PBSC ex-vivo expansion, in particular when poor grafts are obtained.
Assuntos
Antígenos CD34/imunologia , Mobilização de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Mesenquimais/imunologia , Células Cultivadas , Técnicas de Cocultura , Ensaio de Unidades Formadoras de Colônias , Células-Tronco Hematopoéticas/citologia , Humanos , Imunofenotipagem , Interleucina-6/imunologia , Teste de Cultura Mista de Linfócitos , Células-Tronco Mesenquimais/citologiaRESUMO
Obesity is associated with low-grade chronic inflammation. Indeed, adipose tissues (AT) in obese individuals are the former site of progressive infiltration by pro-inflammatory immune cells, which together with increased inflammatory adipokine secretion induce adipocyte insulin resistance. IL-17-producing T (Th17) cells are part of obese AT infiltrating cells, and are likely to be promoted by adipose tissue-derived mesenchymal stem cells, as previously reported by our team. Whereas Th17 cell are physiologically implicated in the neutralization of fungal and bacterial pathogens through activation of neutrophils, they may also play a pivotal role in the onset and/or progression of chronic inflammatory diseases, or cancer, in which obesity is recognized as a risk factor. In this review, we will highlight the pathogenic role of IL-17A producing cells in the mechanisms leading to inflammation in obesity and to progression of obesity-related inflammatory diseases.
RESUMO
BACKGROUND: A bilateral hand allotransplantation was performed in a patient six years ago. Whereas skin is known to be highly immunogenic, grafts have been well accepted up to now. Therefore, here we investigated the putative presence of regulatory T cells in the graft. METHODS: Skin biopsies were performed at different time points and analyzed by immunochemistry. T cells were initially expanded with interleukin (IL)-2. In the latter biopsy, skin was directly analyzed by reverse-transcriptase polymerase chain reaction without any culture. RESULTS: When tested against donor mononuclear cells, donor-primed skin T cells demonstrated unresponsiveness and inhibited donor-directed blood T cell alloresponse. Moreover, their T-cell receptor-Vbeta repertoire was skewed, in contrast to that of peripheral blood T cells. Retrospectively, nuclear FoxP3 expression in skin was measured by immunohistochemistry and was found positive at that time, but appeared to increase with time. This result was supported by the measurement of FoxP3 messenger RNA (mRNA) expression in the latter fresh biopsy, which showed higher levels than that of blood, together with no expression of perforin mRNA, but increased expression of transforming growth factor-beta and IL-10. No FoxP3 mRNA expression was found in the contralateral leg, due to the absence of T cell infiltrate. CONCLUSION: This study shows the presence of the FoxP3 marker, in a well accepted human composite tissue allograft, up to six years posttransplantation. Because a suppressive cytokinic profile was also detected intragraft, in the absence of perforin mRNA expression, our data suggest that regulatory T cells could play a role in the long-term survival of this allograft.